Journal of Molecular and Cellular Researches
Year:2014 | Volume:26 | Issue:3|Papers Count:15

Microglial cells are the smallest glial cells in the central nervous system (CNS) which, as brain macrophages have an important role in regulation of innate immunity. Quick phenotypic changes of these cells from quiescent state to activated shape and secretion of diffusible factors, show their unique ability in response to inflammatory stimuli. Nitric oxide (NO), a free radical, is one of the molecules which produce by activated microglial cells and have a pivotal role in progression of neurodegenerative disorders like Parkinson Disease (PD) and Alzheimer Disease (AD). Therefore, Restrain the overactivation of microglial cells, is a chief strategy to decrease neurotoxic damages which threat central nervous system. In this research we investigate the ability of “IMOD” as an herbal drug, to decrease the bacterial lipopolysaccharide (LPS)-induced NO production in newborn rat brains. The production of NO was assessed by Griess assay whilst the cell viability was determined by MTT assay. Results show that high dilutions of this drug have an anti-inflammatory effect on microglial cells, which is able to decrease the amount of produced NO in these cells. With these results we can use IMOD to decrease neurological damages in neurodegenerative disorders due to microglial activation.

In this experiment, we studied the effects of drought stress under field conditions on chlorophyll a, chlorophyll b, carotenoids, rubisco large subunit (rbsl) and the activities of antioxidant enzymes in barley at early anthesis, watery ripe, late milk and soft dough stages of two genotypes with different yield (Q13: 1390 Kg/ha and Q20:1582 Kg/ha). The results showed that under oxidative stress chlorophyll b is more sensitive than chlorophyll a. The amount of cartenoids increased at initial time of stress period, but declined with the progress of stress. The amount of rbsl declined during senescence and drought stress. The activity of SOD declined with plant age in both water treatments. However it was more in plants under drought stress of Q13. APX activity declined with age in both water treatments of Q13. There were no significant difference with age in Q20 APX activity. Drought stress triggered increases in antioxidant enzymes activities. We concluded that Q20 has more ability to maintain SOD activity during water deficit stress and APX activity during senescence; therefore it seems that this ability is a key role for increasing the yield of Q20 in compare to Q13.

Purple bacteria could use sunlight as an energy source for their growth and reproduction. Since these bacteria are not able to use hydrogen of water as an electron donor, they need to use reduced forms of hydrogen like H2S, H2 or organic matters in which oxygen molecules are not produced in their photosynthesis process. These bacteria are of specified organisms in water found in many water resources. Because of their growth effects on wastewater stabilization ponds which have been considered as common wastewater facilities in Iran, investigations on their growth condition and important affecting parameters have been considered for design and operation of such facilities. The occurrence reasons of purple sulfur bacteria (PSB) in waste stabilization ponds were investigated. Physical and chemical parameters which affect on PSB growth like BOD5, COD, dissolved oxygen (DO), temperature, and hydrogen sulfide (H2S) were observed monthly. Results showed that the PSB dominated in two periods, which the first was in midsummer (in primary facultative pond) and the second was in midwinter (in tertiary facultative pond). This condition was occurred by low DO concentration in facultative ponds due to high organic loading rate (OLR) and also high concentration of H2S which were caused overcoming of the PSB over algae. Results showed that the OLR was the main affecting parameters in the overcoming of PSB.

Cocksfoot (Dactylis glomerata) is a perennial grass that is used for pastures and hay production. This study evaluated total proteins profiles of 110 genotypes of cocksfoot from 11 populations, to determine the extent of genetic diversity. On the basis of SDS-PAGE, 25 reproducible bands were used for analysis and genetic diversity was estimated based on the number of different protein peptides. Molecular weight of bands ranged from 7638 to 2768850 Dalton. The average of polymorphic band over total bands detected ranged from 0.14 (in population Ardebil) to 0.41 (in population Ourmieh). SDS-PAGE of total proteins showed high inter- and intra-population diversity and no clear differentiation on the basis of origin or source. The mean genetic distance among populations was 0.045, ranging from o.017 between Karaj1 and Ardabil to 0.132 between Karaj3 and Pasand2. The correlation between genetic and geographical distance matrices was not significance (R=0.02, p=0.280), analyzed with mantel test, indicating lack of clinal trends in variation of total proteins. These results suggested that the genetic base of cultivated cocksfoot should be broadened by involving diverse parents in the breeding program. Expansion of the genetic base for cocksfoot breeding might be accomplished by systematic use of germplasm that differs in protein profiles and has better quantitative traits.

Shigella and enteroinvasive Escherichia coli (EIEC) strains are belong to the gram negative bacterium family which cause the most communicable of bacterial dysenteries (shigellosis). The virG gene as an essential factor is required for pathogenicity of shigella. This protein encoded via virG gene which located on the outer membrane (OM) of bacteria surface. Furthermore, the VirG protein belonging to autotransporter (AT) protein family of extracellular protein's gram-negative bacteria. This protein interacts with the host actin regulatory protein which caused intra- and intercellular spreading throughout the host epithelium. One approach for construction of Shigella vaccines is to attenuate wild-type strains by mutating genes which regulate specific virulence properties. The aim of this research was identification, cloning, sequencing virG gene and construction of a live attenuated DvirG by use of the l Red recombinase in Shigella dysenteriae type 1isolated from patients with shigellosis. By use of serological and Polymerase Chain Reaction (PCR) tests, species and serotype of shigella seperated from patient was confirmed. According to the Gene Bank database (NCBI), detection primers of virG gene was designed, after amplification of virG, this construct was cloned to pGEM-7zf vector as cloning vector. Finally, sequencing was performed by use of universal primers of vector. The pKD46 as helper plasmid (composed of the essential genes for induce recombination under arabinose promoter) was transformed to the native shigella by using electroporation. After designing the primes which require for induction recombination, the PCR reaction performed through pKD3 vector (which compose of chloramphenicol cassette flanked by FRT sequence). After purification of chloramphenicol cassette, this cassette transformed to the native shigella which carrying pKD46 by using electronic shock. Mutant strain obtained by occurring homologous recombination between chloramphenicol cassette and virG gene. Then, antibiotic cassette was eliminated via FRT sites by application of pCP20 (carrying FLP recombinase) as helper plasmid. Precision of process was confirmed by phenotypic (growth resistant strain to chlramphenicol) and genotypic (PCR reaction with external primers and then sequencing of its product) characterization. The result sequencing of virG in comparison with standard strain as reference strain (Accession No. CP000035) was identical (100%). According to bioinformatics analysis mutant strain, deletion of 3220 bp of virG gene was confirmed. Utilization of l Red recombinase system facilitates mutant construction in more cost-effective method in comparison with the other techniques such as suicide vector.

Nowadays, monoclonal antibodies are the most applicable biological drugs. To that end, the construction of ScFv is the first step, having the advantage of small size, rapid clearance from blood, and better penetration to the tissues. A monoclonal antibody (A1D12) that enhances the activation of plasminogen 3-50-fold in the presence of plasminogen activators is a good candidate as a drug for coronary disorders. The main goal of this study is to construct the single chain fragment of variable (ScFv) for humanization. In this investigation ScFv of A1D12 has constructed and expressed in E.coli, which is capable of recognizing the corresponding antigen according to the results of SDS-PAGE and competitive ELISA respectively.

Firefly luciferase (luc) is one of the most important industrial enzymes which have various applications in medicine, biotechnology and molecular biology. luc is also a suitable candidate both in in vivo bioluminescence imaging and reporter systems, due to having interesting features such as high sensitivity, reproducibility, and being measured quantitatively. In this study, Iranian firefly Lampyris turkestanicus luciferase gene with red emitting light was subcloned and introduced into African violet (Saintpaulia ionantha) plants. The luc gene was isolated by PCR, and was subcloned in pCAMBIA1304 vector. The construct was confirmed by several methods: colony PCR, PCR, digestion, sequencing and BLAST analysis. The Agrobacterium-mediated transformation method was used to introduce luc into African violet genome and putative transgenic plants were selected on the selective medium containing hygromycin and cefotaxim. Finally, transgenic plants were confirmed by PCR, RT-PCR and Luminometric analysis. The results of luminometric assay showed the activity of luciferase enzyme in the leaves of some plants. In this study, the maximum emitted light was 20000 RLU/sec, while in the control plant, the light emission was not detected.

An extremely halophilic archaeon, Haloarcula sp. IRU1 which isolated from Uromia lake can produce carotenoid pigments. In this study, we assayed several methods for pigment extraction by using different solvents, ultimately, the cell breakage by employing relatively soft glass powder and acetone: hexane as solvents was selected as the best method. Then, the pigments were separated by Thin Layer Chromatography. In order to pigment separating, we examined several solvents with different percentages and the best separating was performed with acetone: petroleum ether (35:65; v/v). There were appeared six bands on TLC plate and the first one with the least RF was the most high intensity band. A few carotenoid standards were used for primary identification of pigments. The highest intensity band on TLC plate was determined as the main pigment component in this archaeon and was scraped off from TLC for identification. For pigment analyzing, UV-Visible, FT-IR, Mass Spectrometry and NMR spectrums of this band were taken and compared with present references. These results suggested the main pigment of this archaeon is bacterioruberin which is sterified with acetate group.

The Xanthomonas genus is one of the most important groups of crop plant pathogenic bacteria and they are one of main causal agents of post-harvest spoilage and economical loss. In this research, 71 agricultural soil samples collected from suburb of Ray and Karaj cities. Screening of bacteria on semiselective SX agar medium, performing morphological and biochemical tests and evaluation of two specific virulence factors resulted in a number of X. campestris isolates among all putative growing bacteria. Relative percentage of X. campestris to total number of bacteria grown on SX agar was 0.69%. Capability of exopolysaccharide production by average concentration of 10.98 g l-1 and average viscosity of 1403 cP and presence of xanthomonadin pigment with maximum absorbance index within the range of 441.0-444.7 nm wavelengths, as two main virulence factors, was determined and confirmed. Molecular identification for 25% of the randomly selected isolates possessing virulence factors, indicated X. campestris species by confirming the presence of specific hrcC gene marker. This study indicates that despite lack of any report on prevalence of the related diseases, the potentially virulent X. campestris can be found in soil of cabbage farms in suburb of Ray and Karaj cities. As a case study, existence of these strains in soil is noticeable because of probability of losses caused by spoilage of agricultural products which is prevalent in Iran.

Metalworking fluids (MWFs) are extensively used in the metalworking industry to cool and lubricate the tool work piece interface and protect the work piece from corrosion. The aim of this study was to survey the microbial load of MWFs rate and evaluate the effectiveness of formaldehyde and non formaldehyde biocides on microbial elimination in a molding unit at Sarcheshmeh copper complex. During a period of 9 months, two samples were collected per week from sump of molding unit at Sarcheshmeh copper complex by Grab sampling procedure. Using emulsification, surface tension and HPLC tests, MWFs deterioration were detected. Finally, the efficiency of four selective biocides on dominant microbial populations and biocide efficiency were assessed by HPLC. Among formaldehyde and non formaldehyde biocides, glutaraldehyde had the ability for complete removal of MWFs microbial contamination. Therefore this biocide in a dose higher than 20 ppm is recommended in MWFs.

Today, role of cancer stem cells as a basic and original factor in cancer metastasis is discussed. Cancer stem cells secret different materials such as protease enzymes. Interstitial collagenase or fibroblast collagenase is a member from large family of protease enzymes destroying basic membrane and extracellular matrix. Therefore, it is not only facilitates progression of cancer cells but also having key role in cancer cell viability through releasing of growth and angiogenesis factors. Insertion of a guanine nucleotide at the position of -1607 introduces a binding site for ETS transcriptional factor in the promoter of the gene. Binding ETS to the 2G polymorphism allele would be increased expression of fibroblast collagenase gene which it could be progressed releasing of cancer stem cells, and therefore, it could be facilitated metastasis. Goal of this study is valuation of association between the 2G/1G polymorphism with progression and metastasis of breast cancer, and also patient viability. Blood samples of 200 cases and 100 controls were collected from two capital cities, Tehran and Isfahan; equally. Samples were genotyped using PCR-RFLP method. Statistical analyses show that 2G/2G genotype frequency is much more in cases rather than controls (29.5% compare with 23%). Patients were divided into two groups of with metastatic activity (M+) and without metastatic activity (M-). The 2G/2G genotype was more frequent in M+ group compared with control group (OR=2.03, CI95%=1.05-3.94). In next step, patients without metastasis were followed up for 32 months. Three years total viability analyses in patients who carrying the 2G/2G genotype in comparing with other patients showed no significantly difference. However rate of cancer special viability and viability without disease in the two groups were statistically significant. In conclusion, to our knowledge, the present epidemiological study for the first time indicates that the 2G/2G genotype polymorphism could be a facilitated factor for progression and metastasis of breast cancer and causing of viability reduction in patients.

Probiotics are live microbial supplements with a wide range beneficial in human life.Effect of probiotic microorganisms on human cancer is a controversial issue. In this work the inhibitory effect of the supernatant of an autochthonous isolate of bifidobacterium bifidum on the CacoII cells of human colonic carcinoma was investigated. Different concentration of 100,200, and 300 ml/ml of the supernatant of the probiotic bacteria harvested at 24,48 and 72 hours of bifidobacterial growth in MRS media were applied in to the 96 well microplates each containing 8000 cells of CacoII after neutralization of the pH with 1N NaOH. The percentage of cancer cells inhibition observed ranged from 55% to 82% which obtained from 100 ml/ml (24h) and 300 ml (72h) supernatants. The inhibitory effect of the probiotic bacteria on human cancer cells seems to be concentrated dependent and not affected by neutralization.

The oil spillage has always been a source of contamination in the soil affecting the environment, the plant and animal life. The source of the contamination is usually due to faulty extraction of the oil from the earth, refining, processing and finally the transportation. The spillage of the crude oil can penetrate through the soil and get to the underground water and the farming lands and by doing so, damage the animals grass land and humans farming products. Lentil is a widely used crop in human's diet and so it makes it important to be investigated. In this study, the effect of the contaminated soil on the growth and germination of the lentil and the activity of alkaline phosphatase has been investigated. The results showed that the 5% concentration of oil in the soil delayed and decreased the number of germination and the biomass of the shoots and the roots, also decreasing the normal size of the leaves .Therefore 5% concentration of crude oil in the soil showed it is a suitable dose to study the effect of contamination on the enzyme. The results showed that the activity of the enzyme in the roots of the control was significantly different compared with that of the contaminated ones. Measurement of the enzyme kinetics for the determination of the Vmax and Km also showed a significant difference, suggesting that there may be an alkaline phasphatase isozyme presence in the roots, which is responsible for this difference observed between the contaminated and the control samples.

The aim of this study was a strain-improvement program for Trichoderma reesei PTCC 5142 by using a combination of UV light and NTG (N-methyl-N'-nitro-N-nitrosoguanidine) for enhanced cellulase production. Following mutagenesis after several rounds, mutant A6: 2 was selected from a total of 6500 colonies. Results obtained after 4 days were: Enzyme activity 1.26 U/ml and 0.82 U/ml for exoglucanase and endoglucanase, respectively. The comparative results showed increased production exoglucanase and endoglucanase by mutant A6: 2 than Trichoderma reesei PTCC 5142 to amount 130% for exoglucanase and 156% for endoglucanase.

Transgenic expression of hairpins can induce RNA silencing pathways by means of dsRNA with sequence homology to a plant mRNA. Using transgenic tobacco plants (N. tabacum cv. Samsun NN) in which the RDR1 gene was silenced to different extents by a hairpin structure homologous to the tobacco RDR1 gene, we evaluated the stability of gene silencing, by analyzing the RDR1 gene expression levels and the plant susceptibility to systemic infection by PVYo. The second (T2) and third (T3) generations of RDR1 silenced transgenic lines were tested by semi-quantitative RT-PCR to evaluate the expression levels of defense-related genes including IVR, ERF5, AOX1, and RDR6, one week after PVYo infection. The results showed that T2 transgenic tobacco lines transcribed the RDR1 and the other defense-related genes to a lower level than the corresponding T3 transgenic lines. The T2 lines were more susceptible to PVYo infection than the T3 lines and the RDR1 gene was expressed to the same level in the T3 lines as in non-transformed tobacco. The efficiency of hairpin structure-mediated silencing of the RDR1 gene decreased through different generations. As the RDR1-regulated RDR6 is required for the spreading of silencing throughout the plant, we propose that suppressing RDR1 leads to inhibition of RDR6, which then leads to loss of silencing by the RDR1 hairpin transgene after vertical gene transmission.